I am interested in the molecular mechanism of X inactivation, and of its spreading in the autosomal segment of X-autosome translocations. To study these phenomena at the molecular level I will isolate chromosome X- and autosome-specific DNA fragments from a recombinant DNA library constructed from flow-sorted chromosomes. The chromosome-specific DNA fragments will then be used as probes to study changes in methylation and chromatin structure during inactivation of the mouse Cattanach X-autosome translocation (which consists of the insertion of a piece of chromosome 7 in the chromosome X, resulting in the formation of chromosome X(7)). A series of hybridization experiments will be used to identify and isolate chromosome-specific DNA fragments from the A phage library enriched in chromosome X(7) material. To identify X-specific DNA I will use a mouse x hamster hybrid cell line which retains only the mouse X chromosome. The level of hybridization of the DNA segment to male and female mouse DNA will be compared. To identify 7-specific DNA I will use mice carrying a deletion in the piece of chromosome 7 involved in the Cattanach translocation. Two different types of blot hybridization experiments will be used to study changes in DNA methylation and chromatin conformation during X inactivation. To study methylation changes I will digest DNA from embryonic and germ cells containing active or inactive X(7) chromosomes with two related endonucleases. Msp I and Hpa II, which differ in their ability to cut the sequence CCGG when it is methylated. To study changes in chromatin conformation during X inactivation I will examine the DNase I sensitivity of X- and 7-specific sequences in cells containing active or inactive X(7) chromosomes.